1. Field of the Invention
The present invention is directed to an enhanced regeneration system for cereal plants, especially wheat and barley, and the use thereof in genetic engineering.
2. Description of the Background Art
The crop species belonging to family Gramineae (Poaceae), order Graminale, class Monocotyledoneae, and subdivision Angiospermae of the Plant Kingdom are known as cereals. The major cereal grain crops of the world include crops such as wheat, rice, barley, corn, oats, rye, sorghum, and millets. Cereal grain crops contribute about 90% of the total grain production of the world. Among cereal grain crops wheat, rice, and corn provide three-fourth of the world's cereal grain production. Barley, sorghum, rye, oats and millets account for the remainder (Stoskopf, N.C. 1985, Cereal Grain Crops, Virginia, U.S.A.). Cereals are regarded as principal source of carbohydrate and protein in animal and human diet. In addition, cereals provide fats, minerals and vitamins. The starch stored in cereal grains can also be fermented into ethanol for use in beverages and as a fuel source.
Both wheat and barley occupy a unique position in the global agricultural economy because of their widespread cultivation and trade at the international level. The annual world production of wheat and barley for 1992 is estimated to be 547 and 171 million tons, respectively (Market Commentary, 1991, Agricultural Canada Publication). Wheat and barley together accounts for more than 40% of the total world's cereal grain production. Wheat is the single largest commodity traded in the world. Wheat and barley together accounts for more than 50% of the total world grain export. With the constantly increasing world population, the demand for wheat, barley and other cereals is expected to rise, resulting in an increase in production of about 2% annually (Stoskopf, 1985).
Conventional plant breeding has, thus far, contributed significantly to the improvement of cereal crops. However, the advent of genetic engineering techniques now provides an opportunity for further improvement of wheat, barley, and other cereals, by incorporating genes for resistance to herbicides, insect pests and diseases, and better nutritional quality into elite genotypes.
A major problem with genetic engineering of wheat and barley is the inability to recover fertile plants from the transformed cells. Although several procedures have been described in the literature for plant regeneration from various tissues of wheat and barley, all of these procedures have drawbacks.
Many different types of explants, such as mature and immature zygotic embryos, immature inflorescence segments, anthers, young leaves and roots have been successfully used for establishing in vitro cultures of both wheat and barley (Vasil, 1988; Gobel and Lorz, 1988). Plants have also been regenerated from such cultures via organogenesis and somatic embryogenesis. (Organogenesis refers to development of adventitious shoots or roots (unipolar structures) which maintain their link to initial explant tissue or callus originated from explant tissues. Somatic embryogenesis refers to development of distinct somatic embryos, (bipolar structures) with shoot and root apices integrated into one axis, from somatic cells. The somatic embryos are not attached to the parental tissue and are capable of germinating into complete plants.) However, in general, the regeneration frequency from most explants has been very low. Moreover, the success with plant regeneration from the most responsive explants such as immature zygotic embryos and inflorescence segments (Thomas and Scott, 1985; Redway et al., 1990) depends on the tedious and subjective process of identification, selection and maintenance of embryogenic callus. The entire process of shoot regeneration from these explants often takes more than ten weeks from the initiation of cultures. This prolonged culture period not only results in loss of regeneration potential, but also adds to the risk of genetic instability among regenerants. The major limitation with the use of immature anther culture for plant regeneration is the genotype dependence of the process and the occurrence of albino plantlets at a high frequency among regenerants.
In the recent past, several attempts have been made to establish cell suspension cultures from embryogenic callus cultures and subsequent plant regeneration from established cell suspensions or protoplasts isolated from such cultures, in both wheat and barley (Vasil, 1990; Jahne et al., 1991; He et al., 1992). However, in most cases either the procedure was not reproducible or it resulted in the production of infertile plants, with the exception of one instance in which normal fertile plants were recovered from protoplasts of an Australian genotype of wheat (He et al., 1992). Additionally, the establishment of cell suspension cultures of wheat and barley is an extremely difficult and time consuming process. Consequently, the immature zygotic embryos have been extensively used for obtaining embryogenic callus and subsequent plant regeneration in wheat and barley. Since immature zygotic embryos contain embryo axes, the shoot regeneration from such explants is sometimes confused with the precocious germination of embryo axis or axillary shoot proliferation from the remains of embryo axis removed after germination. The precocious germination of embryo axis is undesirable for genetic engineering and other biotechnological applications of tissue culture methods and therefore should be avoided. The most desirable mode of plant regeneration from somatic cells is through somatic embryogenesis as described in this invention.
The process of shoot regeneration from intact immature zygotic embryos is slow because a large proportion of callus produced from such explants constitutes an undesirable non-embryogenic callus. The non-embryogenic callus grows at a faster rate than embryogenic callus and thus suppresses the growth of embryogenic callus by competing for nutrients and other constituents of the tissue culture medium. The identification, selection and maintenance of embryogenic callus from the mixture of different callus types is as mentioned earlier, a tedious and time consuming process.
Successful genetic engineering of cereal crop plants primarily depends upon the availability of a high frequency, genotype-independent regeneration procedure which has the potential of producing a large number of plants in a short period of time.